Scanning Ion Conductance Microscopy: A new approach to understanding cancer cell stiffness

扫描离子电导显微镜：了解癌细胞硬度的新方法

• 批准号: 2439276
• 金额: --
• 依托单位: St George's, University of London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2439276
• 关键词: Scanning; Ion; Conductance; Microscopy; new

The UK incidence of melanoma (skin cancer) has increased by 45% in the past decade. Melanoma is the 5th most common cancer in the UK with around 16,000 new melanoma skin cancer cases each year. However, mortality rates of melanoma in the UK have increased by 156% since the early 1970s. While an increase in incidence may be due to the more proficient clinical screening techniques, there has been little improvement in the understanding of melanoma development, prognosis and treatments. Pigmentation within the skin strongly protects against ultraviolet (UV) light-induced DNA damage. In human skin, melanin is produced by melanocytes which are present in the basal epidermis and hair follicles. Melanin within the cell acts as an UV light filter and form barriers to prevent UV light causing damage to DNA. Alterations and mutations within genes in melanocyte development and important metabolic pathways can alter the cells growth and proliferation leading to cancer formation. Melanocyte transformation into cancer and melanoma cell transformation into aggressive metastatic melanoma is associated with alterations in the mechanical properties of the cell. It is hypothesised that metastatic cells are softer than their non metastatic counterpart to allow the cell to travel in the bloodstream to secondary sites in the body and survive there. However, in the case for melanoma, this relationship is not clear as there have been studies which show that highly metastatic cell lines are stiffer than their non metastatic counterpart. In addition, a higher level of ROS has been seen in almost all cancers and is associated with tumour progression. The tumour metabolic environment influences growth and treatment response and can therefore ensure the cancer cells survive in their micro-environment and metastasise. The relationship between cancer cell stiffness and ROS is not yet known. If cell stiffness does play an important role in cancer metastasis, perhaps ROS also has a part to play in cytoskeletal alterations to allow for metastasis. Currently, the most widely used method for mapping cell stiffness is atomic force microscopy. Our group has developed a new method which is non-contact and low-force so that no damage occurs to the cell when scanning. The method is based upon scanning ion conductance microscopy (SICM) which can map cell topography with nanoscale precision. This project is the first to assess the correlation between cell stiffness and metastatic ability in melanoma cell line using established immortal melanocyte cell lines from the Functional Genomics Cell Bank at St George's. This project meets the MRC's interdisciplinary core skill as it relies upon skills in:- Biophysics - use of the SICM cell stiffness equipment (through collaboration with Imperial College London and ICAPPIC Limited)- Microscopy - SICM, fluorescence and confocal microscopy- Cell Biology - mammalian cell culture, cell proliferation assays, cell viability assays, ROS degradation assays- Molecular Biology - immunostaining, immunoblotting- Statistics - processing and analysing datasets- Presentation - presenting findings in the form of presentations, posters and peer review publicationsThis project also encompasses the MRC's quantitative core skills as there will be processing and analysis of interdisciplinary datasets. The range of known skills (molecular biology) and newly developed skills (biophysics, microscopy and cell culture) used throughout this PhD will allow the role of cell stiffness within melanoma to be understood and allow quantitative, accurate 3D mapping of cell stiffness during different stages of melanoma development and progression. The SICM system could also have a future diagnostic application in distinguishing between cancer and non-cancer cells in patients and monitor key biomarkers to optimise treatment. Keywords - Cancer, Melanoma, Cell Biology, Molecular Biology, Biomarker, Cell Stiffness, Mechanobiology

英国黑色素瘤（皮肤癌）的发病率在过去十年中增加了45%。黑色素瘤是英国第五大常见癌症，每年约有16，000例新的黑色素瘤皮肤癌病例。然而，自20世纪70年代初以来，英国黑素瘤的死亡率增加了156%。虽然发病率的增加可能是由于更熟练的临床筛查技术，但对黑色素瘤发展，预后和治疗的理解几乎没有改善。皮肤内的色素沉着强烈地保护免受紫外线（UV）光诱导的DNA损伤。在人类皮肤中，黑色素由存在于基底表皮和毛囊中的黑色素细胞产生。细胞内的黑色素充当紫外线过滤器，并形成屏障以防止紫外线对DNA造成损伤。黑素细胞发育和重要代谢途径中基因的改变和突变可以改变细胞生长和增殖，导致癌症形成。黑素细胞转化为癌症和黑色素瘤细胞转化为侵袭性转移性黑色素瘤与细胞机械性质的改变有关。据推测，转移性细胞比非转移性细胞更柔软，以允许细胞在血流中移动到身体的次要部位并在那里存活。然而，在黑色素瘤的情况下，这种关系并不清楚，因为已经有研究表明，高转移性细胞系比其非转移性对应物更硬。此外，在几乎所有癌症中都观察到较高水平的ROS，并且与肿瘤进展相关。肿瘤代谢环境影响生长和治疗反应，因此可以确保癌细胞在其微环境中存活并转移。癌细胞硬度和ROS之间的关系尚不清楚。如果细胞硬度确实在癌症转移中发挥重要作用，那么ROS可能也在细胞骨架改变中发挥作用，从而允许转移。目前，最广泛使用的映射细胞刚度的方法是原子力显微镜。我们的团队开发了一种新的方法，它是非接触和低力的，因此在扫描时不会对细胞造成损伤。该方法是基于扫描离子电导显微镜（SICM），可以映射细胞形貌与纳米级精度。该项目是第一个使用来自St乔治功能基因组细胞库的已建立的永生黑素细胞系评估黑色素瘤细胞系中细胞硬度和转移能力之间的相关性的项目。该项目符合MRC的跨学科核心技能，因为它依赖于以下技能：- 生物物理学-使用SICM细胞硬度设备（通过与帝国理工学院伦敦和ICAPPIC有限公司合作）-显微镜- SICM，荧光和共聚焦显微镜-细胞生物学-哺乳动物细胞培养，细胞增殖测定，细胞活力测定，ROS降解测定-分子生物学-免疫染色，免疫印迹-统计学-处理和分析数据集-演示文稿-以演示文稿的形式展示研究结果，海报和同行评审出版物这个项目还包括MRC的定量核心技能，因为将有跨学科的处理和分析，数据集。已知的技能（分子生物学）和新开发的技能（生物物理学，显微镜和细胞培养）在整个博士学位的范围将允许黑色素瘤内的细胞硬度的作用被理解，并允许在黑色素瘤的发展和进展的不同阶段的细胞硬度的定量，准确的3D映射。SICM系统还可以在未来的诊断应用中区分患者的癌症和非癌细胞，并监测关键的生物标志物以优化治疗。关键词-癌症，黑色素瘤，细胞生物学，分子生物学，生物标志物，细胞硬度，机械生物学